Members of the Cys-loop family of transmitter-gated ion channels are pentameric membrane proteins that respond to the binding of transmitter with a conformational change resulting in the opening of a gate and an increased affinity to the agonist. The muscle-type nicotinic acetylcholine receptor (nAChR) has served as a prototype in Cys-loop ion channel research due to its large single-channel conductance resulting in a high signal-to-noise ratio, and the relatively simple kinetic properties. In the nAChR, the two transmitter binding sites are located in the extracellular domain of the receptor at the interfaces between an a and a d subunit, and an a and an e. The subunit in the fifth position (b) is not directly involved in transmitter binding. The focus of this proposal is the non-transmitter-binding b subunit. We will use a combination of molecular biological, electrophysiological, and kinetic analyses approaches to determine the energetics and timing of gating motions in: i) the second membrane-spanning domain, ii) the extracellular region, and iii) the region comprising the linker between the second and third membrane-spanning regions and the pre-TM1 region. The work will elucidate the involvement of the b subunit in the conformational changes that lead to channel activation, and provide insight into the sources of molecular motions in the non- transmitter-binding subunit. The completion of the proposed work will increase our understanding of how transmitter-gated ion channels function, and how the non-transmitter-binding subunit shapes receptor function. PUBLIC HEALTH RELEVANCE: The nicotinic acetylcholine receptor mediates fast synaptic transmission in the vertebrate neuromuscular junction. The pentameric protein contains two transmitter binding sites located at subunit-subunit interfaces. This project is aimed at elucidating the role of the fifth subunit, which is not involved in transmitter binding, in the structural motions resulting in channel gating.